GSM6774224: 010-RTr16-EfficacyW16D1; Macaca mulatta; RNA-Seq
Blood was collected at specified time points post 1st MVA immunization into PAXgene Blood RNA tubes (BD Biosciences) and the RNA was extracted using the PAXgene Blood RNA Kit IVD (Qiagen). RNA quality was assessed using an Agilent Bioanalyzer and then one microgram of total RNA was subjected to globin transcript depletion using the GLOBINclear Kit, human (ThermoFisher Scientific). Ten nanograms of the globin-depleted RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio), which uses oligo-dT priming for the reverse transcription step. This process would yield low recovery of non-coding RNAs and hence we focused our analysis on protein coding transcripts. Amplified cDNA was fragmented and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina). Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled at equimolar concentrations, and sequenced on an Illumina HiSeq3000 at 100SR, yielding 25-30 million reads per sample.